The present invention relates to the interaction of transcription factor E2F with ubiquitin, and to assays for modulators of this interaction.
E2F transcription factors control the expression of at least three groups of genes that are involved in cell cycle regulation. First, E2F sites have been found in the promoter of the immediate early gene c-myc (Hiebert et al., 1989; Oswald et al., 1994). In addition, E2F contributes to the regulation of several genes whose expression is activated in the G1 phase of the cell cycle, including cyclin E, E2F-1 and p107 (Degregori et al., 1995; Johnson et al., 1994; Neuman et al., 1994; Zhu et al., 1995). Finally, E2F contributes to the cell cycle-regulated expression of a number of genes that are required during S phase, such as cyclin A, dihydrofolate reductase, DNA polymerase a and thymidine kinase (reviewed by Farnham et al., 1993). E2F transcription factors are heterodimers that contain one of five related E2F polypeptides and one of two DP polypeptides (reviewed by Beijersbergen and Bernards, 1996).
The activity of the various E2F transcription factors is regulated at three different levels. First, the abundance of E2F is regulated at the level of transcription. For example, E2F-4 is the most prominent E2F species in quiescent cells, whereas E2F-1 is absent from quiescent cells and is transcriptionally induced in late G1 after serum stimulation (Johnson et al., 1994; Sardet et al., 1995). Second, transactivation by E2Fs is negatively regulated by complex formation with one of three members of the retinoblastoma pocket protein family, pRb, p107 and p130 (reviewed by Beijersbergen and Bernards, 1996). E2F-1, 2, and 3 interact preferentially with pRb; E2F-4 with p107 and p130 (Beijersbergen et al., 1994; Ginsberg et al., 1994; Vairo et al., 1995); E2F-5 with p130 only (Hijmans et al., 1995). These E2F-pocket protein complexes are likely to perform different functions during the cell cycle as the timing of their appearance differs. Most quiescent cells have one major E2F complex that consists of E2F-4 in complex with p130 (Chittenden et al., 1993; Cobrinik et al., 1993; Vairo et al., 1995). Exponentially growing cells contain significant amounts of free E2F and E2F-p107 complexes (Beijersbergen et al., 1995; Cobrinik et al., 1993; Lees et al., 1992; Shirodkar et al., 1992). E2F-pocket protein complexes are regulated by phosphorylation of the pocket proteins by G1 cyclin/cyclin-dependent kinase (cdk) complexes. pRb can be phosphorylated by cyclin D/cdk4, cyclin E/cdk2 and cyclin A/cdk2 kinase complexes (Dowdy et al., 1993; Ewen et al., 1993; Hinds et al., 1992). In contrast, p107 is only efficiently phosphorylated by cyclin D/cdk4 (Beijersbergen et al., 1995). In addition, several viral oncoproteins, including adenovirus E1A, can disrupt E2F-pocket protein complexes through high affinity binding to the pocket proteins (Whyte et al., 1988). A third level of regulation of E2F activity concerns the regulation of DNA binding activity. Krek et al. (1994) have shown that E2F-1 can interact directly with cyclin A, which results in phosphorylation of DP-1 in S phase, causing down-regulation of E2F DNA binding activity. Down-regulation of E2F in S phase appears to be important in cellular homeostasis, as over-expression of E2F, or mutants of E2F that resist cyclin A down-regulation, can cause apoptosis and transformation (Beijersbergen et al., 1994; Johnson et al., 1994; Krek et al., 1995; Qin et al., 1994; Singh et al., 1994; Wu and Levine, 1994).
E2F DNA binding sites in promoters can act both as positive and negative regulatory elements, depending on the promoter context (Lam and Watson, 1993). The action of E2F sites as negative regulatory elements is most readily explained by the finding that pocket proteins can mediate active transcriptional Thus, E2F/pocket protein complexes found in quiescent cells may contribute to maintaining quiescence through active transcriptional silencing of growth factor-activated genes.
Many of the proteins that contribute to regulation of the cell cycle appear and disappear rapidly, often in a cell cycle-regulated manner. Degradation of unstable proteins frequently involves the ubiquitin-proteasome pathway (Hilt and Wolf, 1996; Hochstrasser, 1995; Jentsch, 1992; Jentsch and Schlenker, 1995; Rubin and Finley, 1995). This system acts by covalent attachment of multiple ubiquitin polypeptides to the substrate. Ubiquitin is a highly conserved 76 amino acid protein found in eukaryotic cells. Ubiquitination requires the action of three different enzymes. A ubiquitin-activating enzyme (E1), which binds ubiquitin and transfers it to an ubiquitin conjugating enzyme (UBC or E2) (Haas and Rose, 1982; Pickart and Rose, 1985), which in turn may need the assistance of a ubiquitin ligase (E3) to attach the ubiquitin residue covalently to the substrate at a lysine residue. Each ubiquitin covalently attached to a lysine residue of the substrate protein is further ubiquitinated at a lysine residue in the ubiquitin sequence itself. Multi-ubiquitination acts as a sorting signal which targets substrates for rapid degradation by the proteasome, a complex of proteolytic enzymes (Chau et al., 1989).
An important link between the ubiquitin-proteasome machinery and cell cycle regulation came from the finding that CDC34, a yeast gene required for the G1 to S transition, was identical to yeast UBC3, a ubiquitin conjugating enzyme (Goebl et al., 1994). Other substrates of Cdc34 include yeast G1 cyclins and the cyclin/cdk inhibitor p40sic1 (Deshaies et al., 1995; Schwob et al., 1994; Yaglom et al., 1995). In mammalian cells, the p27 cyclin/cdk inhibitor was recently shown to be a degraded in a cell cycle dependent fashion by the ubiquitin-proteasome pathway (Pagano et al., 1995). In budding yeast, the S-phase cyclin Clb5 and the mitotic cyclin Clb2 are ubiquitinated through UBC9 (Seufert et al., 1995) and Xenopus mitotic cyclins have also been shown to be degraded through ubiquitination (Glotzer et al., 1991).
We report here that E2F transcription factors are unstable due to destruction by the ubiquitin-proteasome pathway and that their degradation is highly regulated.
Accordingly, the present invention provides an assay method for an inhibitor of transcription factor E2F ubiquitin-mediated degradation which method comprises:
a) bringing a polypeptide which contains a domain which renders E2F a substrate for ubiquitination into contact with a candidate inhibitor; and
b) determining whether or not the candidate inhibitor is capable of reducing ubiquintination of said polypeptide.
The polypeptide may be an E2F protein or fragment thereof which is capable of binding to a pocket protein or the polypeptide may comprise an indicator polypeptide such as LacZ.
Preferably, the domain which renders E2F a substrate for ubiquitination comprises the 63 amino acid C-terminal region of E2F-l, or the 138 amino acid C-terminal domain of E2F-4, or portion thereof such as the 112 C-terminal amino acids.
The assay will normally be conducted in a format where the polypeptide is expressed in a host cell from a recombinant expression vector, such as an expression vector where the polypeptide is operably linked to a CMV promoter.
In one embodiment, a DP-1 polypeptide is co-expressed in the host cell.
Assays of the invention may be conducted in the presence of a proteasome inhibitor.
In the assay of the invention the determining of whether or not the candidate inhibitor is capable of reducing ubiquintination of said polypeptide may be performed by providing ubiquitin and determining the amount of said ubiquitin which has been bound to said polypeptide. This may be achieved by ubiquitin or contains an expression vector capable of expressing ubiquitin. The ubiquitin may be tagged with an epitope capable of binding to a monoclonal antibody, such as an HA epitope.
In another aspect, the invention also provides a vector which comprises a nucleotide sequence encoding a truncated E2F polypeptide wherein said truncated polypeptide does not contain a C-terminal domain capable of ubiquitination. One such polypeptide is E2F-1 1-374.
In a further aspect, the invention also provides an assay method for an enhancer of transcription factor E2F ubiquitin-mediated degradation which method comprises:
a) bringing polypeptide which contains a domain which renders E2F a substrate for ubiquitination into contact with a candidate promoter; and
b) determining whether or not the candidate promoter is capable of enhancing ubiquintination of said polypeptide.
The various preferred embodiments of the assay for inhibitors described herein may also be used in this further aspect of the invention.
The amino acid sequences of E2F-1 and E2F-4 are shown as SEQ. ID NO.1 and SEQ. ID No.4 respectively.
In the present application, reference to transcription factor E2F refers to the family of transcription factors capable of forming a heterodimer with a DP-transcription factor as described by Beijerbergen and Bernards, 1996, (the disclosure of which is incorporated herein by reference) and capable of forming a complex with a member of the retinoblastoma pocket protein family, such as pRb, p107 or p130.
E2F proteins are found in mammalian cells. Representative of specific members of the E2F family are those members found in human cells. For example, human E2F-1 is shown as SEQ ID NO. 1. Human E2F-2 and E2F-3 are disclosed in, for example, Lees et al (1993) Mol.Cell.Biol 13; 7813-7825, the disclosure of which is incorporated herein by reference. Human E2F-4 is shown as SEQ ID NO. 3. Human E2F-5 is disclosed in, for example, WO/96/25494, the disclosure of which is incorporated herein by reference.
E2F proteins from other species, such as mice, are also available or may be cloned using standard methodology known in the art per se and by reference to the techniques used to clone the human E2F proteins mentioned above.
Synthetic variants of the human or other species E2F proteins may be used. Synthetic variants include those which have at least 80%, preferably at least 90%, homology to human E2F-1 or E2F-4. More preferably such variants correspond to the sequence of said human E2F-l or E2F-4 but have one or more, e.g. from 1 to 20, such as from 2, 5 or 10 substitutions, deletions or insertions of amino acids.
Such variants will desirably retain one or both of the following functional properties:
(i) the ability to cause the E2F protein to undergo ubiquitin-mediated degradation as described herein for the various specific E2F proteins exemplified; and
(ii) the ability to bind to a pocket protein.
Both functional properties may be determined by routine experimentation by reference to methods illustrated in the accompanying examples.
The assay method for modulators of E2F ubiquitin-mediated degradation is useful in determining candidate substances which can influence the progression of the cell cycle. This is because E2F is required in cell cycle control and its modulation may influence progression or arrest of the cycle or may induce apoptosis. Thus modulator substances which are capable of reducing or increasing ubiquitination of E2F will be useful in arresting the cell cycle. Such substances will be useful in research, for example in providing synchronous populations of example in the treatment of proliferative diseases such as cancer.
The assay may be conducted in any suitable format designed to examine the interaction of E2F with ubiquitin. For example, the assay could be conducted in an in vitro cell-free system in which the polypeptide, inhibitor substance and ubiquitin are provided together with other cellular components involved in the ubiquitination process. A rabbit reticulocyte lysate may be used as the basis for a cell free system, supplemented with a ubiquitin activating enzyme (E1) and ATP.
Preferably however the assay is conducted in a host cell which contains an expression vector capable of expressing the E2F polypeptide. The host cell may also contain a further expression vector capable of expressing a DP-polypeptide such as DP-1. The host cell may also contain a vector capable of expressing ubiquitin. Any or all of the polypeptides encoded by the expression vectors may include an epitope which is capable of being detected by a monoclonal antibody. The epitope is generally one which is not normally present in the host cell such as the HA epitope of influenza virus.
Where a vector encoding a ubiquitin is supplied to the assay of the invention, the ubiquitin encoded by the vector may be any suitable ubiquitin compatible with the host cell or in vitro assay system being used. For example, various mammalian ubiquitins have been characterised and their sequences may be obtained by reference to the published literature or databases. As indicated above, the ubiquitin may be tagged with an epitope detectable by an antibody and this epitope may be fused to the N- or C- terminal of the ubiquitin.
Generally, naturally occuring sequences of ubiquitin will be used, for example mammalian, preferably human, although minor modifications to the sequences may be made, for example during the engineering of nucleic acid encoding ubiquitin into a sequence encoding an epitope some residues may be altered or deleted. An example of a vector encoding ubiquitin may be found in Treier et al, 1994, the disclosure of which is incorporated herein by reference.
The host cell may be any suitable host cell in which ubiquitination of E2F can occur. The suitability of host cells may be determined by repeating procedures analogous to those described in accompanying Examples in a potential host cell and determining whether ubiquitination of E2F occurs. Suitable host cells include mammalian host cells such as human or murine host cells. Other host cells include insect or yeast host cells.
Although a naturally-occurring E2F protein or synthetic variant thereof may be used the assay may be conducted with other polypeptides such as fusion proteins which retain the domain of E2F which renders E2F a substrate for ubiquitination. We have found this domain is present in the C-terminal region of E2F. The experiments described in the accompanying Examples demonstrate that this region is contained within the 63 C-terminal amino acids of E2F-1 although smaller portions of this region may also be used provided they retain their substrate function. Thus for example a fragment of this 63 amino acid region comprising 10, 20, 30, 40 or 50 amino acids may be used provided such a fragment retains the ability to render E2F a substrate for ubiquitination. The fragment may be a C-terminal fragment of the 63 amino acid region or an internal fragment. The ability of such fragments to function as a substrate for ubiquitination may be tested by routine methods based on the accompanying examples or by attaching the fragment to an indicator protein as described below.
Similarly, we have found that the 138 C-terminal region of E2F-4 may be used, and fragments of this, also of for example 10, 20, 30, 40, 50, 100, 112 or 120 amino acids may be used as the ubiquitination domain. Again, such fragments may be internal or C-terminal.
The polypeptides used in the assay methods of the invention may contain more than one ubiquitination domain, for example from 1 to 10 such as 2, 3, 4, or 5 domains. These domains may be present in tandem repeats or in both the N- and C-terminal regions of the polypeptide.
Alternatively, the ubiquitination domain may be attached to an indicator protein such as protein which can provide a colour change when the necessary substrates are added to the cell. Usually such attachment is by preparing a fusion protein encoded by nucleic acid which links sequence encoding the domain to sequence encoding the indicator protein, the linkage being in-frame. This may be accomplished by routine cloning techniques. The domain may be N- or C-terminal to the indicator protein. Such indicator proteins include lacZ or horseradish peroxidase. In this embodiment of the assay the effect of the candidate inhibitor may be measured by adding the necessary colour change substrates in the presence and absence of the candidate and measuring the amount of colour change produced.
Alternatively, the indicator polypeptide can be a sequence specific transcription activator and the assay can include a reporter construct which is expressed in the presence of the sequence specific transcription activator. Such activators include, for example, VP16 activator of herpes virus or the GAL4 activator of yeast.
Where the assay of the invention is conducted in a host cell and the various components of the assay are provided on expression vectors the expression vectors may use any suitable promoter capable of functioning in the host cell. We have used promoters from CMV although other viral or cellular promoters may be used. It is however desirable that the promoter is not regulated by E2F and is expressed independently of the cell cycle.
Where the polypeptide used in the assay of the invention is an E2F protein capable of binding to a pocket protein the pocket protein is desirably pRb, p107 or p130.
The assay may be conducted in the presence of a proteasome inhibitor such as Cbz-LLL. Where such inhibitor is added poly-ubiquitination of the polypeptide will occur and can be detected in large quantities since polypeptide-ubiquitin conjugates will accumulate in the cell and not be subject degradation in the proteasome.
Although it is preferred that the assay of the invention is used to examine inhibitors of E2F ubiquitin-mediated degradation the assay is also suitable for examining promoters of such degradation. The various preferred embodiments of the assay described above may also be used in the assay for candidate promoter substances.
The nature and amount of candidate inhibitor or promoter substances which will be used in the assay will depend on the assay format being used and the nature of the substance. These can be determined by routine trial and error by those of ordinary skill in the art. Candidate substances include peptides based on the C-terminal region of E2F which may compete with native E2F for ubiquitin in a cell. Where the assay of the invention is conducted in a host cell the amount of candidate substance used may depend on its ability to enter the cell. Methods and adjuvants for enhancing the permeability of cells are known in the art and may be used in assays of the present invention. Anywhere between sub-nanomolar to micromolar concentrations of candidate substances may be used, for example from 1 nM to 100 xcexcM.
Thus assays of the invention may be used as the basis for rational drug design based on peptides or mimics thereof designed around the sequence of the ubiquitination domain or fragments thereof such as those described above.
Antibodies directed against the ubiquitination domain, or fragments of such antibodies which retain the ability to bind this domain (including humanized antibodies) may be used in the diagnosis, prognosis or treatment of conditions in which abberant mutations or other loss of function in the ubiquitination domain of E2F. Such conditions will generally be those involving uncontrolled cell proliferation such as cancer.
Antibodies or fragments thereof directed against this domain, antibodies or fragments thereof against ubiquitin, and compounds which bind an E2F/DP protein heterodimer may be used singly or in combination in assays for or methods of treating uncontrolled cell proliferation.
In a separate aspect, the present invention provides vectors which encode a truncated E2F (such as E2F-1, E2F-2, E2F-3, E2F-4 or E2F-5) which do not contain a C-terminal domain capable of ubiquitination. Such polypeptides include truncated E2F-1 or E2F-4 comprising residues 1 to about 400, for example about 300, 320, 350, 375, 390, 410 or 420. The N-terminal may also be truncated by a few amino acids, for example about 5, 10, 15 or amino acids. One such polypeptide is E2F-1 1-374. Another is E2F-4 1-301. Other truncated E2F polypeptides such as those mentioned above may be made and tested in accordance with the methods described in the accompanying Examples. Alternatively, the E2F may contain a C-terminal region but that region may be modified to prevent ubiquitination of the polypeptide in a host cell, for example by internal deletion of the ubiquitination fi domain. The invention also provides vectors which encode a fusion protein of an indicator polypeptide and one or more ubiquitination domains of E2F, such polypeptides being as described above.
These truncated and modified polypeptides will be useful in delivering transcription factor E2F to a cell which is resistant to ubiquitination. Such polypeptides may thus reduce cellular proliferation since they will accumulate in a cell and mediate transcriptional repression via their interaction with pocket proteins.
Vectors encoding the various truncated and modified E2F polypeptides as well as other polypeptides described above may in the art. The accompanying Examples describe a number of suitable vectors and further vectors can be made from these by routine methods. For example, truncated or modified polypeptides may be made by site-directed mutagenesis of nucleic acid encoding the unmodified proteins. PCR cloning techniques may be used to obtain nucleic acid encoding other transcription factors not specifically cited in the Examples and these techniques can also be used to introduce changes to the naturally occurring DNA sequence in order to produce truncated or modified polypeptides.
Generally vectors for use in the invention will be DNA vectors although RNA vectors may also be used.